Tissue transglutaminase-2 (TGase-2) has been suggested to be involved in a number of vitally important biological responses ranging from neuronal differentiation and neurodegenerative diseases to the regulation of cell growth and various cancer-associated activities including extracellular matrix modulation and metastasis. However, thus far, relatively little is known regarding the molecular basis by which TGase-2 participates in this broad array of cellular and biological processes. What makes this protein especially interesting is that it is capable of GTP-binding and hydrolytic activities like signaling G-proteins, as well as an enzymatic cross-linking (transamidation) activity that catalyzes the covalent linkages between glutamine residues and primary amino groups, resulting in the formation of new protein-protein and protein-polyamine complexes. During the past funding period, we have found that these different functions enable TGase-2 to play an essential part in ensuring the survival of various cells against apoptotic challenges, as well as having a key role in the EGF-stimulated anchorage-independent growth, migration, and invasive activity of cancer cells. In this renewal application, we propose to build on these different findings to learn more about the EGF-dependent signaling pathways that regulate TGase-2 expression and function, as well as the mechanisms by which TGase-2 contributes to EGF-stimulated cell growth and migration. This will entail the following lines of investigation. 1) Understanding the role of TGase-2 in the anchorage-independent growth of breast cancer cells. Here we will set out to determine how EGF stimulates the up-regulation of TGase-2 in breast cancer cell lines and how this enhances their ability to grow in the absence of a substratum. 2) Understanding the role of TGase-2 in cell migration. These studies will identify the signals used by EGF to direct the recruitment of TGase-2 to the plasma membrane surface and the leading edges of cells, and determine why TGase-2 is necessary for EGF- stimulated cell migration. 3) Understanding how TGase-2 is secreted from cells and why this provides protection against apoptotic challenges. This line of study stems from our findings that secreted/extracellular TGase-2 plays an important role in protecting cells against apoptosis-inducing stimuli. We will determine the mechanism by which TGase-2 is secreted from cells, how this provides a survival advantage, and whether this also accounts for the role played by TGase-2 in EGF-stimulated invasive activity. By understanding the mechanisms of action and regulation of TGase-2, we expect to gain new insights into fundamentally important cellular signaling activities responsible for cell survival and growth factor-stimulated cell migration. These studies should also provide us with a better understanding of how the de-regulation of this interesting protein can contribute to various pathological conditions such as the development of metastatic cancers. PUBLIC HEALTH RELEVANCE Tissue transglutaminase is an interesting multi-functional protein that has been implicated in making cancer cells resistant to chemotherapeutic drugs as well as in promoting the metastatic potential of these cells. It also has been linked to neurodegenerative disorders, especially Alzheimer's disease. Thus, by understanding how tissue transglutaminase is regulated and performs its normal functions in cells, and what events lead to its inappropriate actions, we hope to obtain information that will shed new light on the development of malignant cancers and possibly other disease states and degenerative disorders. The expectation is that this information will highlight novel targets and strategies for therapeutic intervention.